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Genome STRIP test installation error

susancsusanc Member
edited January 2013 in GenomeSTRiP

I just installed Genome STRIP and ran the 2 test scripts provided. The 2nd script, genotyping.sh, failed after 1.5 mins with the error appended below. It looks like I should run 'bwa aln' again, but with what parameters? All suggestions appreciated.....

INFO 13:34:56,460 HelpFormatter - The Genome Analysis Toolkit (GATK) v1.0.5039M, Compiled 2011/01/20 22:58:34
INFO 13:34:56,461 HelpFormatter - Copyright (c) 2010 The Broad Institute
INFO 13:34:56,461 HelpFormatter - Please view our documentation at http://www.broadinstitute.org/gsa/wiki
INFO 13:34:56,461 HelpFormatter - For support, please view our support site at http://getsatisfaction.com/gsa
INFO 13:34:56,461 HelpFormatter - Program Args: -T SVAltAligner -R /usr/local/gatk/svtoolkit/installtest/data/human_b36_chr1.fasta -I /usr/local/gatk/svtoolkit/installtest/data/installtest.bam -O /usr/local/gatk/svtoolkit/installtest/test2/altalign/installtest.alt.bam -md test2/metadata -U ALLOW_UNINDEXED_BAM -altReference test2/altalign/installtest.sites.alt.fasta -alignUnmappedMates true
INFO 13:34:56,461 HelpFormatter - Date/Time: 2012/08/14 13:34:56
INFO 13:34:56,462 HelpFormatter - ---------------------------------------------------------------------------
INFO 13:34:56,462 HelpFormatter - ---------------------------------------------------------------------------
INFO 13:34:56,466 GenomeAnalysisEngine - Strictness is SILENT
INFO 13:34:57,226 GATKRunReport - Aggregating data for run report

ERROR ------------------------------------------------------------------------------------------
ERROR stack trace

org.broadinstitute.sting.utils.exceptions.ReviewedStingException: Reverse BWT file is missing; please rerun 'bwa aln' to regenerate it.
at org.broadinstitute.sting.alignment.bwa.c.BWACAligner.(BWACAligner.java:39)
at org.broadinstitute.sv.breakpoint.BreakpointAligner.initialize(BreakpointAligner.java:46)
at org.broadinstitute.sv.breakpoint.SVAltAlignerWalker.initialize(SVAltAlignerWalker.java:86)
at org.broadinstitute.sting.gatk.executive.LinearMicroScheduler.execute(LinearMicroScheduler.java:49)
at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.execute(GenomeAnalysisEngine.java:217)
at org.broadinstitute.sting.gatk.CommandLineExecutable.execute(CommandLineExecutable.java:111)
at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:239)
at org.broadinstitute.sting.gatk.CommandLineGATK.main(CommandLineGATK.java:87)

ERROR ------------------------------------------------------------------------------------------
ERROR A GATK RUNTIME ERROR has occurred (version 1.0.5039M):
ERROR
ERROR Please visit to wiki to see if this is a known problem
ERROR If not, please post the error, with stack trace, to the GATK forum
ERROR Visit our wiki for extensive documentation http://www.broadinstitute.org/gsa/wiki
ERROR Visit our forum to view answers to commonly asked questions http://getsatisfaction.com/gsa
ERROR
ERROR MESSAGE: Reverse BWT file is missing; please rerun 'bwa aln' to regenerate it.
ERROR ------------------------------------------------------------------------------------------
Post edited by Geraldine_VdAuwera on

Best Answer

  • bhandsakerbhandsaker admin
    Accepted Answer

    Hi, Susan,
    I suspect there was an error in the job run just prior to this one, which should have created the bwa index.
    It is also possible that bwa failed without returning a non-zero error code, so it looks like the job succeeded.
    Can you check the log file for the last few jobs, especially for one that ran "bwa index" and post the output?
    And if the problem isn't obvious, can you send me a directory listing for "ls -l test2/altalign" where you ran the installtest?
    Also useful would be for you to run "bwa" with no arguments and let me know which version you have installed.

Answers

  • bhandsakerbhandsaker Member, Broadie, Moderator admin
    Accepted Answer

    Hi, Susan,
    I suspect there was an error in the job run just prior to this one, which should have created the bwa index.
    It is also possible that bwa failed without returning a non-zero error code, so it looks like the job succeeded.
    Can you check the log file for the last few jobs, especially for one that ran "bwa index" and post the output?
    And if the problem isn't obvious, can you send me a directory listing for "ls -l test2/altalign" where you ran the installtest?
    Also useful would be for you to run "bwa" with no arguments and let me know which version you have installed.

  • susancsusanc Member

    Ah, the problem was with that we had a local version of BWA 0.6.2-r126 installed, which was being used by the script. When I set my path to use the bwa 0.5.8c (r1536) that was packaged with Genome Strip, it worked fine. Thanks very much.

  • bhandsakerbhandsaker Member, Broadie, Moderator admin

    OK, thanks, that's good to know. Do you have the log file from the failed run so I can see why the newer version of bwa failed?

  • susancsusanc Member
    edited August 2012

    I can't see anything obvious in the logs from the failed run, but perhaps you will be able to.

    Here is the BWA run:

    --- logs/[email protected]
    [bwa_index] Pack FASTA... 0.00 sec
    [bwa_index] Construct BWT for the packed sequence...
    [bwa_index] 0.00 seconds elapse.
    [bwa_index] Update BWT... 0.00 sec
    [bwa_index] Pack forward-only FASTA... 0.00 sec
    [bwa_index] Construct SA from BWT and Occ... 0.00 sec
    [main] Version: 0.6.2-r126
    [main] CMD: bwa index test2/altalign/installtest.sites.alt.fasta
    [main] Real time: 0.006 sec; CPU: 0.002 sec
    -------------------------------------------
    

    Here is the log from the next step, also from the failed run:

    ------ logs/[email protected]
    Failed to run job.
    Command line:
    sh /usr/local/gatk/svtoolkit/installtest/tmpdir/.exec2344906357378266364
    Exit code: 1
    Standard error contained: 
    INFO  13:34:56,458 HelpFormatter - --------------------------------------------------------------------------- 
    INFO  13:34:56,460 HelpFormatter - The Genome Analysis Toolkit (GATK) v1.0.5039M, Compiled 2011/01/20 22:58:34 
    INFO  13:34:56,461 HelpFormatter - Copyright (c) 2010 The Broad Institute 
    INFO  13:34:56,461 HelpFormatter - Please view our documentation at http://www.broadinstitute.org/gsa/wiki 
    INFO  13:34:56,461 HelpFormatter - For support, please view our support site at http://getsatisfaction.com/gsa 
    INFO  13:34:56,461 HelpFormatter - Program Args: -T SVAltAligner -R /usr/local/gatk/svtoolkit/installtest/data/human_b36_chr1.fasta -I /usr/
    local/gatk/svtoolkit/installtest/data/installtest.bam -O /usr/local/gatk/svtoolkit/installtest/test2/altalign/installtest.alt.bam -md test2/
    metadata -U ALLOW_UNINDEXED_BAM -altReference test2/altalign/installtest.sites.alt.fasta -alignUnmappedMates true 
    INFO  13:34:56,461 HelpFormatter - Date/Time: 2012/08/14 13:34:56 
    INFO  13:34:56,462 HelpFormatter - --------------------------------------------------------------------------- 
    INFO  13:34:56,462 HelpFormatter - --------------------------------------------------------------------------- 
    INFO  13:34:56,466 GenomeAnalysisEngine - Strictness is SILENT 
    INFO  13:34:57,226 GATKRunReport - Aggregating data for run report 
    ##### ERROR ------------------------------------------------------------------------------------------
    ##### ERROR stack trace 
    org.broadinstitute.sting.utils.exceptions.ReviewedStingException: Reverse BWT file is missing; please rerun 'bwa aln' to regenerate it.
            at org.broadinstitute.sting.alignment.bwa.c.BWACAligner.<init>(BWACAligner.java:39)
            at org.broadinstitute.sv.breakpoint.BreakpointAligner.initialize(BreakpointAligner.java:46)
            at org.broadinstitute.sv.breakpoint.SVAltAlignerWalker.initialize(SVAltAlignerWalker.java:86)
            at org.broadinstitute.sting.gatk.executive.LinearMicroScheduler.execute(LinearMicroScheduler.java:49)
            at org.broadinstitute.sting.gatk.GenomeAnalysisEngine.execute(GenomeAnalysisEngine.java:217)
            at org.broadinstitute.sting.gatk.CommandLineExecutable.execute(CommandLineExecutable.java:111)
            at org.broadinstitute.sting.commandline.CommandLineProgram.start(CommandLineProgram.java:239)
            at org.broadinstitute.sting.gatk.CommandLineGATK.main(CommandLineGATK.java:87)
    ##### ERROR ------------------------------------------------------------------------------------------
    ##### ERROR A GATK RUNTIME ERROR has occurred (version 1.0.5039M):
    ##### ERROR
    ##### ERROR Please visit to wiki to see if this is a known problem
    ##### ERROR If not, please post the error, with stack trace, to the GATK forum
    ##### ERROR Visit our wiki for extensive documentation http://www.broadinstitute.org/gsa/wiki
    ##### ERROR Visit our forum to view answers to commonly asked questions http://getsatisfaction.com/gsa
    ##### ERROR
    ##### ERROR MESSAGE: Reverse BWT file is missing; please rerun 'bwa aln' to regenerate it.
    ##### ERROR ------------------------------------------------------------------------------------------
    
    -------------------------------------------
    
  • susancsusanc Member

    p.s. apologies for the poor formatting.

  • Geraldine_VdAuweraGeraldine_VdAuwera Cambridge, MAMember, Administrator, Broadie admin

    Tip for formatting large code blocks: use a texteditor to add tabs (or 4 spaces) at the beginning of each line. In TextWrangler (on Mac) for example if you select all the text and hit cmd-] it will automatically indent the whole block. All good text editors (like Notepad++ on Windows) have similar capabilities. Then when you copy the resulting indented text into your post or comment, it will be formatted properly.

  • bhandsakerbhandsaker Member, Broadie, Moderator admin

    Thanks, Susan. I'll check it out and see why the newer version of bwa is failing.

  • reedwarblerreedwarbler Member

    hi,I met the same problem, even change to bwa 0.5.8c (r1536), have you solve this problem?

  • bhandsakerbhandsaker Member, Broadie, Moderator admin

    You should make sure that you removed all intermediate files so that the compatible version of bwa (0.5.8c) is the one indexing the alt allele sequences. You can verify this by looking in the log files to see which version is being run.

  • jfarrelljfarrell Member ✭✭

    I ran into this issue when trying to run the ComputeGenomeMask tool with the latest release.

    The issue is related to the change in the index files generated with bwa 0.6 which comes with GenomeSTRiP.

    The newer version of bwa generate five files (.amb, .ann, .bwt, .pac, .sa files) but with bwa-0.5.9 there are nine index files (.fai, .rpac, .amb, .ann, .pac, .bwt, .rbwt, .rsa, .sa nine files).

    ComputeGenomeMask is complaining the some of those index files are missing and exits.

    So this tool looks like it needs to be updated to use the new bwa 6.0 index file structure being generated.

    From the bwa web site.....

    Since version 0.6, BWA has been able to work with a reference genome longer than 4GB. This feature makes it possible to integrate the forward and reverse complemented genome in one FM-index, which speeds up both BWA-short and BWA-SW. As a tradeoff, BWA uses more memory because it has to keep all positions and ranks in 64-bit integers, twice larger than 32-bit integers used in the previous versions.

  • bhandsakerbhandsaker Member, Broadie, Moderator admin

    That's correct. We bundle an older version of bwa 0.5 with Genome STRiP and you need to use this version to index the reference file before running ComputeGenomeMask. We use the bwa C API internally so upgrading for us is a little more work than it might seem.

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